We have continued our studies of chromatin structure in the neighborhood of expressed genes. The globin gene family in chicken erythroid cells serves as a model system in which it is possible to study the mechanisms associated with regulation of the individual members of the family during erythroid development. We have extended our studies of the way in which hypersensitive sites are generated in the chicken beta globin enhancer. We constructed plasmids on which we can control both replication and the generation of hypersensitive sites dependent on expression of the erythroid factor GATA-1. We showed that hypersensitive site generation does not require replication, and that the site generated is nucleosome-free and different from that obtained in vitro. In other studies, we have solved the structure of the complex between the DNA binding domain of the GAGA protein,which is implicated in disruption of chromatin structure, and its target site on DNA. The structure reveals that binding stability arises from a combination of contacts of alpha helices in the DNA major groove and of an extended basic polypeptide chain in the minor groove, a motif also found in the GATA-1 DNA binding domain. In other work, we have shown that the N-terminal domains of GATA-2 and -3, unlike that of GATA-1, is able to bind independently to DNA, with a preference for the sequence GATC. Binding is aided by two basic regions on either side of the zinc finger, a property that may help distinguish the binding properties of these two proteins from those of GATA-1. We have also extended studies of the 1.2 kb insulator DNA sequence at the 5' end of the chicken beta-globin locus by showing that a smaller fragment, about 250 bp in length, carries a large part of the activity. Experiments with the full length insulator fragment and smaller fragments show that they are capable of directional blocking of enhancer activity in transient expression assays as well as in stable transformation experiments, thus making it easier to study the mechanism of insulation.